1. Field of the Invention
The present invention is related to a fluorescence detecting method. More particularly, the present invention is related to a fluorescence detecting method that utilizes surface plasmon.
2. Description of the Related Art
Fluorometry is conventionally used in for biological measurements and the like, as an easy and highly sensitive measuring method. In fluorometry, a sample, which is considered to contain a detection target substance that emits fluorescence when excited by light having a specific wavelength, is irradiated with an excitation light beam of the aforementioned specific wavelength. The presence of the detection target substance can be confirmed by detecting the fluorescence due to the excitation. In the case that the detection target substance is not a fluorescent substance, a substance labeled by a fluorescent substance that specifically bonds with the detection target substance is caused to contact the sample. Thereafter, fluorescence is detected in the same manner as described above, thereby confirming the presence of the bond, that is, the detection target substance.
With recent advances in the performance of photodetectors, such as cooled CCD's, fluorometry has become indispensable in biological research. In addition, fluorescent pigments having fluorescence quantum yields that exceed 0.2, which is the standard for practical use, such as FITC (fluorescence: 525 nm, fluorescence quantum yield: 0.6) and Cy5 (fluorescence: 680 nm, fluorescence quantum yield: 0.3) have been developed as fluorescent labeling materials and are being widely used.
Further, high sensitivity detection on the order of 1 pM and less is being realized, by amplifying fluorescence signals employing electric field enhancing fields due to surface plasmon, as described in Japanese Patent Application No. 2006-255374 and by M. M. L. M. Vareiro et al., “Surface Plasmon Fluorescence Measurements of Human Chorionic Gonadotrophin: Role of Antibody Orientation in Obtaining Enhanced Sensitivity and Limit of Detection”, Analytical Chemistry, Vol. 77, No. 8, pp. 2426-2431, 2005. This method is referred to as surface plasmon electric field enhanced fluorescent spectroscopy.
Raman spectroscopy using a metal probe which is capable of detecting a substance at the molecular level, as described in Y. Inouye and S. Kawata, “Near field Raman spectroscopy and imaging using a tip enhanced field”, Spectral Researches, Vol. 51, No. 6, pp. 276-285, 2002. In this Raman spectroscopy method, light is caused to enter the leading end of a metal probe, to cause local plasmon to be generated. A localized strong electric field which is generated by the local plasmon between the probe and a substrate is utilized. Thereby, the scattering cross sectional area during the Raman process by molecules directly beneath the probe is effectively increased. Theoretically, it is considered that an increased intensity from ten times to 106 times the intensity of incident light can be obtained (refer to section 2.2.1 at page 277 of Y. Inouye and S. Kawata, “Near field Raman spectroscopy and imaging using a tip enhanced field”, Spectral Researches, Vol. 51, No. 6, 2002).
However, Raman signals are greatly influenced by the environment and conditions in which detection target substances are placed, such as solvents. In addition, vibrations of foreign substances are also reflected in spectra. Therefore, although Raman spectroscopy is effective for qualitative analysis, expectations cannot be held regarding the quantitative properties thereof. Further, Raman spectroscopy apparatuses are generally large, expensive, and have poor operability.